Synthesis of dextran and dextran analogues of predetermined molecular weight



Nov. 22, 1955 DEXTRAN YIELD PERCENT OF THEORY DEXTRAN YIELD, PERCENT OF THEORY H. M. TSUCHIYA ETAL 2,724,679 SYNTHESIS OF DEXTRAN AND DEXTRAN ANALOGUES OF PREDETERMINED MOLECULAR WEIGHT Filed May 12, 1953 IOOF- I II I I 40 I,

I [I] 2 o T l O 'I l I I I I I l I I I I 34 38 42 46 5O 54 58 62 66 7O ETHANOL, PERCENT IOOP- O l l l I I I I l 36 4O 44 48 52 56 6O 64 68 v 72 METHANOL, PERCENT HENRY M. TSUCHIYA NISON N. HELLMAN HAROLD J. KOEPSELL United States Patent SYNTHESIS OF DEXTRAN AND DEXTRAN ANA- OF PREDETERMINED MOLECULAR Henry M. Tsuchiya and Nison N. Hellman, Peoria, 111., and Harold J. Koepsell, Grosse Pointe, Mich., assignors to the United States of Americaas represented by the "Secretary of Agriculture r r Application May 12, 1953, Serial No, 354,664

11 Claims. 01. l95 -3-1) (Granted under Title 35, U. S. Code (1952), see. 266) A non-exclusive, irrevocable, royalty-free license in the invention herein described, f for all governmental purposes, throughout theworld, with the power to grant sub-licenses for such purposes, is hereby granted to the Government of the United States of America.

This invention relates to the synthesis of polysaccharides by means of dextransucrase and associated enzymes by methods involving the control of molecular growth so that the molecular weight of the productrnay be predetermined. The process of the invention isbasically an enzymatic synthesis, i. e., enzymatic conversion of sucrose and certain other sugars to polysaccharides.

The products produced are carbohydrate gums that may be called dextrans or dextran-like analogues. They differ from previously known dextrans produced by direct 276,035, filed March 11, 1952. As disclosed and claimed in that application such polysaccharide products are of a relativelylow molecular weight. The average molecular weight of the polymer formed may be controlled to some degree by employing predetermined concentrations and ratios of sucrose toalternate glucosyl acceptors,

which may be sugars or sugar derivatives. In this specification the alternate glucosyl acceptors are also termed primers. The average molecular weight of the .polymers prepared according to .that invention mayvary from about 4,000 to about 20,000 depending upon the .concentrationsand ratios of sucrose to alternate glucosyl .acceptors. The products resulting from tho -build up ,of

glucosyl groups upon the accepting molecule may be termed isomaltodextran, maltodextran, etc according as the alternate glucosyl acceptoris isomaltose, maltose, etc.

We have also determined that low molecular weight dextran is produced when the enzymatic synthesis, or polymerization, is carriedout at higher initial levels of sucrose concentration as disclosed in tour :co-pending application, Serial No. 276,033, filed March 11, 1952. By employing predefined initial sucrose concentrations the average-mo1ecular weight of the polymer formed may be controlled within certain limits. In general, theuse of initially high sucrose concentrations leads to the formation principally .of low molecular Weight products; the use of initially low sucrose concentrationslleads .to,.the formation principally of high molecular weight products.

When dextran is synthesized using conventional initial sucrose concentrations of approximately m -percent and activities of dextransucrase as "obtained by 2,724,679 Betented Nov. 22, 1955 processes such as disclosed in patent application, Serial No. 256,586, filed Nov. 15, 1951 by Tsuchiya and Koepsell, and in the absence of added alternate glucosyl acceptors, the molecular Weight of the major product isolated is greater than 1 million, although it may vary somewhat with the reaction conditions employed.

Small amounts of dextran with low molecular weight can be isolated from such reaction mixtures, similar to the dextran with low molecular weight obtained in large amounts in reactions conducted at higher initial sucrose concentrations. Experience has shown that a dextran possessing an average molecular weight of 50,000 to 100,000 is suitable as a blood volume expander. In all of the above mentioned processes the synthesis of major amounts of dextran-like products in the molecular weight range of 50,000 to one million is not accomplished. Ifit is desired to produce ,dextran with average molecular weight of about 75,000, such high molecular weight dextran of more than one million is conventionallydegraded by acid hydrolysis, pyrolysis, enzymatic .degradation or by ultrasonic treatment.

We have now determined some of the factors responsible for the difficulty in the progressive build up of polysaccharide chains to intermediate molecular weights, i. e., 50,000 to one million, in those processes in which the products of dextran synthesis has hitherto been restricted to molecular weights lower than 50,000. We have discovered for example that fructose when present in the reaction mixture in the concentrations inherently resulting in dextran synthesis tends to act tohinder molecular growth. Also we have discovered, for example, that the molecular weight of the alternate glucosyl acceptors, among other things, determines its ability to direct and control the average molecular weight of the polymer formed.

We have now developed simple procedures for the synthesis of dextran and its analogues of predetermined molecular weight by employing dextran-like products. of relatively low molecular weights as alternate glucosyl acceptors, or primers, in the synthesis reaction involving the three essential components, namelydextransucrase, sucrose and the alternate glucosyl acceptor. According to this invention we employ as primer a glucose 'polymer such as low molecular Weight dextran, .e. g. maltodextran and the like, of molecular weight within the range of 5000 to 30,000. We recognize that the nature and concentrations of all three reactants in the synthesis reaction,

i. e., primer, sucrose, and enzyme affect the molecular weight and yield of the products formed. Furthermore, we recognize that the changing concentrations .of the reactants during the synthesis may aifect the polymolecularity of the product formed.

In addition, other reaction variables such .as pH and temperature of the reaction may also affect the yield of dextran of desired molecular weight. The enzymatic synthesis maybe conductedin the pH range of approximately 4.0 to 7.0, but we prefer ,to carryout the polymerization in the range of 4.5 to 5.575. The synthetic reaction may be conducted in the temperature range ,of approximately 4 .C. to 37 .0 However, .we preter to carryout-the synthesis in therange of 10 .C. to 20 0. Although the synthesisproceeds more rapidly at higher temperatures, we have .found that the lower tempera- .tures favor the formation .of intermediate molecular weight polysaccharides and minimize the formation of the higher molecular weight dextran.

Our preferred primer is a low molecular weight dextran of approximately 10,000 to 15,000. Such low molecular weight primer may be produced when tl e enzymatic polymerization is carried out at high levels of sucrose concentration as disclosed in our err-pending ap plication, SerialtNo. 276,033, iiled March ll, 2952. We

tation from the enzymatic polymerization mixture when the polymerization is carried out in this process. Furthermore, we may also employ other polysaccharides as primers, as for example, the polysaccharides obtained by degradation of dextran, starch, glycogen, and other polyglucoses linked through the alpha-glycosidic linkages.

As the molecular weight desired for the product increases above 30,000 the ratios of the concentrations of primer to sucrose will decrease. It may vary for a particular primer from an initial valueof l to 5 to l to 100, the former leading to dextrans of lower molecular weight than the latter. If the primer itself is extremely polymolecular it will cause polymolecularity in the dextran I product. The concentration of the primer may decrease during the reaction as a consequence of participating in the synthesis reaction. However, dextran of such low molecular weight as to render it suitable for primer action may also be formed during the course of reaction.

In the synthesis reaction the dextransucrase enzyme employed in the present invention may be prepared in accordance with the method disclosed in applications, Serial No. 215,623 and 256,586 filed by Koepsell, Kazenko, Ieanes, Sharpe and Wilham and by Tsuchiya and Koepsell, respectively. The enzyme may be used in concentrated or isolated forms, or more conveniently the culture filtrate remaining after removal of the bacterial cells may be used. Moreover, the culture liquor may be used without removal of the bacterial cells, although it is preferred inthat event that the cells will have been renderedinactive under the conditions of our synthesis reaction. The concentration of the enzymes in the polymerization aifects the time required for complete reaction and also the molecular weight and yield and polymolecularity of the product. The preferred concentration of dextransucrase is about 30 to 100 units per milliliter of synthesis mixture, although ranges of 20 to 400 units per milliliter afford satisfactory results. The dextran sucrase concentration may be considerably lower than 20 units per milliliter, as illustrated in Example 7, where, in the first of the three runs, 16,000 units were employed in a liter of synthesis mixturef Inasmuch as the culture filtrate rich in synthesizing enzymes may contain varying amounts of endogenous primers or dextran degrading, or modifying, enzymes which can produce primers during the course of reaction, different sources of dextransucrase may require slight adjustments in the reaction conditions in order to form products of desired molecular weight. However, in our process We prefer that the enzyme concentration not be altered to any great extent as a means for controlling the molecular weight of the product even though variation in the enzyme concentration will affect the molecular weight of the product.

The concentration of sucrose in the reaction may vary over wide limits, from lower than 1 percent to as high as 20; percent. Higher sucrose concentrations induce the formation of lower molecular weight products, probably as a consequence of endogenous primer production and the increasing endogenous fructose concentration. In the operation of the process a higher sucrose concentration can be compensated for by using a lower primer to sucrose ratio. The sucrose concentration may be permitted to decrease continuously during the synthesis, the sugar being added initially to reaction mixtures and being acted upon by the enzymes. Alternatively, the sucrose concentration may be maintained at constant levels during the course of the reaction by the use of dialysis procedures or by the continuous addition of sucrose during the course of the reaction. The degree to which the sucrose concentration is held constant, or in fixed ratio to the alternate glucosyl acceptor and dextransucrase concentrations may control polymolecularity of the dextran product. We prefer to operate our process at initial 10 percent sucrose concentration, adiust the primer to sucrose concentration according to the desired molecular weight of the product, and allow the concentration of sucrose and primer to decrease in the normal fashion during the course of the reaction as they are converted to dextran.

EXAMPLE 1 Dextransucrase enzyme and maltodextran were admixed in solutions of the concentration indicated in the table below. Onehundred ml. portions of these solutions were adjusted to about pH 5.0 and placed in dialysis membrane sacks. The sacks were then placed in 900 ml. of l-percent sucrose solution and the solutions agitated at 25 C. for 48 hours. During this period fructose, which was formed inside the sacks, dialyzed to the outer solution, and sucrose dialyzed from the outer solution to the solution inside the sacks. The resulting products were evaluated by centrifugal fractionation, and the molecular weights determined as listed in Table I. Analytical ultracentrifugal results showed that the reaction mixtures contained high and low molecular weight components separated by a wide gap in sedimentation rates. Centrifugation of reaction mixture at 40,000 g. for 4 hoursseparated the high and low molecular weight components.

The maltodextran, employed as glucosyl acceptor, was prepared inaccordance with our copending application, first mentioned previously. It possessed a molecular weight of about 8,000. Each product produced from this maltodextran exhibited a very sharp and narrow molecular weight range, being even sharper than the molecular weight range obtained by repeated fractionation of hydrolyzed dextran.

Table I Dextran- Malto- N sucrase, dextran, Molecular Yield, 0 units/ g./100 weight grams ml. ml.

*Inherent viscosity determined at relative viscosity of approximately EXAMPLE 2 Table I1 Dextran- Sucro- NO sucrase, dextran, i Molecular Yield, units/100 g./100 weight grams ml. ml.

Yield of low molecular weight product negligible. Molecular Weight given is that of high molecular weight component.

EXAMPLE 3 .The procedure oflExample 1 was repeatedexceptthat the concentration of dextransucrase was T 200 nil. solutions ro'f frucwse Maud enzyme were added slowlyconcentrated sucrose iSOlfltlOl'lS ISO that the sucrose was almost instantaneously converted is idextran and fructose. The :dextransucrase concentration was sufi ficiently high, and rt'he sucroseisolntion added very slowly, dropw'ise overa hour period, so :that the sucrose 6011- centration *was essentially :zero =at-1all times. essentially complete and instantaneous conve'rsionxof sucrose duringthe iculture course of the synthesis :was ascertained by reducing power measurements: uFurfthenmore, the

"concentrations of the sucrose solutions were suc'h that if the sucrose were completelyvantl :instantaneouslyacom verted during the course of. addition, she -fructose concentra'tions in the reaction mixtures were- Jmaintained tattthe desired levels throughout the experiment. Theupl-l was "held at 1510 by the also of 0201M iacetate. ibufier in both the initial 'fructose, enzyme solutions sand in the 'sucrose solutions which were :added. The reactions .werewondncted "at 25 C; and the solutions-agitated mechanicaIly.

The fructose concentration in reaction mixture .A was ..1held at 2.5 percent, inreaction mixture .B cat 75 percent,

'and in reaction: mixture C at percent :throughout the course of the experiment. iEachamixtureacontained84g000 dextransucrase units. To reaction mixture-LA was added =1 800-mls. of a 4.75 percent sucrose solution, to reaction B was added 1800 mls. of a 915 percent lsucrose solution, and *to reaction mixture C was added 31800 imls. of a 195 percent sucrose s'oluti'on. t

At the termination of the synthesisareactions *the apparent yields and the approximate molecular -we'ights of the dextrans produced were determined by fra'etional ethanolprecipitation. To mil. aliqn'otsnof reaction mixtures, warmed to approximately 45 C ='lwere added graded amounts of alcohoL. After these solutions had stood at (lifor-fio hours they were-centrifuged, and

epolarimetri'c measurements were made for ithewnottpreafllplifltd dextran-in thewsupernatannliqnors. flyrdifierence from the amount of total dextran present ;&the amount of: dextran precipitated .at each ethanol. concen- -tration was determined. The :results were .caloulat'ed son the basis that enzymaticalLy synthesized :NRRL

B-512 dextran has a [(1:1 of +201degrees1in-water .sand (of +215 degrees in formamide. wThe rresults1 are .vShOWnrln Figure 1.. --In general, .ihigmmolecular we ght 1,000,000) dextran precipitates at relatively l low methanol or ethanol concentrations 40%) in convaried as shown arc ers trast to low molecular weight polymer which precipitates at higher alcohol levels.

iExamination of the precipitation curves shown in Fig- .ure ,1 indicates that as the fructose concentrations in the :reaction mixtures were increased the formation of high molecular weight dextran fraction was decreased with a concomitant increase in the formation of low molecular :EXAMPLE 6 enzymatic synthesis mixture was prepared as follows: Dextransucrase concentrate from the culture filtrate of a Leuconostoc mesenteroides fermentation and ,an aqueous :solution of maltodextran having an average .molecular weight of about 15,000 was made up to 800 ml. with water, and 200 ml. of a SO- ercent (w./v.) sucrose solution was added dropwise so that g. of sucrose was added. The ratio of the weight of primer to the total weight of sucrose added was 1:10. The amount of dextransucrase employed for each gram of sucrose was 320 units. The pH of the synthesis mixture was held at 5.0 with. 0.01M acetate buffer. The temperature at which the reaction was conducted was 24 C. Toluene was used to prevent the growth of contaminating bacteria. The reaction was permitted to proceed until the sucrose contentbecame minimal. The reaction mixture was centrifuged at 40,000, .g. for 4 hours to separate the .high and low molecular weight components. The components were .purified by two precipitations from methanol. The average molecular Weight of the separated components was determined by light scattering. The above was repeated three itmes under identical conditions with the exception that the quantity of maltodextran primer wasreduced by half each time giving primer to sucrose weight ratios of .1 :20, 1:40, and 1:80, respectively.

Table V High molecular weight Low molecular weight fraction traction ZPPimeFSuGI'OSe Y1 1d Y m i e 1e Molecular Molecular percent percent theoretical eight theoretical Welght 24. 5 235 10 77. 4 73, 500 18. 8 244x10 72. 5 123, 000 34. 1 267x10 67. 7 490, 000 29. 4 274X10 40. 2 1, 150, 000

EXAMPLE 7 The sprocedure of Example 6 was repeated using the .same type. of rnaltodextran as primer but varying the amount of dextransucrase. A primer to total sucrose ,weight -I3IlQz0f 1:10 was employed. The reaction was performed .at dextransucrase concentration of units per gram tofsucrose added and then repeated at 320 units rand zat 640 units vpergramof sucrose added. The ,pHwof -the mixtures it and the temperature at which the reactions were conducted were essentially similar. to those in Ex ampleo. Toluenewas also used.

until the sucrose content became minimal.

'tained at pH 5.0 by 0.01M acetate buffer.

7 EXAMPLE 8" The procedure of Example 7 was repeated using the same type of maltodextran as primer but varying the manner of addition of sucrose. A primer to total sucrose weight ratio of 1:10, and a dextransucrase concentration of 320 units per gram of total sucrose was employed.

Table VII Low molecular v m 53 Weight fraction Mode of sucrose addition fweitght rac ion Molecular yield Y1eld weight Instantaneous 11. 6 e5. 4 75, 900 Dropwise: Over 4-hour period 12. 3 76. 6 45, 600 Over 8-hour period 6. 9 67. 3 56, 100 Over 32'hour period 15. 61 2 45, 400

EXAMPLE 9 An enzymatic synthesis mixture was prepared as follows: To 33.5 gallons of a clarified culture liquor assaying 50 dextransucrase units per ml. from a Leuconoitoo mesenteroides fermentation were added 33.3 pounds sucrose and 3.3 pounds maltodextran'primer similar to that used in Example 6. The final volume of the reaction mixture was 40 gallons. The ph of the reaction mixture was adjusted to pH 5.0. The reaction was conducted at 13 C. Toluene was used as in Example 6. The reaction was permitted to proceed The dextran synthesized was recovered by methanol precipitation at 60 percent. The precipitated material was redissolved in Water to give a dextran concentration of5 percent. The" dextran thus obtained was fractionated by reprecipitating again between 42 and 50 percent methanol. The yield of dextran obtained was 3.9 pounds and the average molecular weight determined by light scattering was EXAMPLE Two 400 ml. reaction mixtures containing 16,000 dextransucrase units, 6 grams of primer (low molecular weight dextran fraction obtained by fractional methanol precipitation from a previously conducted enzymatic polymerization carried out under conditions essentially similar to the present experiment), and 40 grams of sucrose were prepared. The pH of solutions were main- Contaminating bacterial growth was inhibited by the addition of small amounts of toluene to the reaction mixtures. Reaction mixture A was held at 30 C. and reaction mixture B at 15 C. for 8 hours. At the termination of the experiment enzymatic activity in these reaction mixtures.

was stopped by heating at 100 C.

The apparent yields and approximate molecular Weights of dextrans synthesized were determined by tractional solvent precipitation in a manner essentially similar to that described in Example 5. Instead of ethanol, methanol was used as the precipitating agent. The results are shown in Figure 2.

Examination of Figure 2 shows that the formation of the high molecular weight dextran fraction was suppressed with concomitant increased formation of poly-..

saccharides with intermediate molecular weight by lowering the temperature of the reaction from 30 C. to 15 C. Past experience has shown that dextran with methanol precipitation characteristics of the intermediate molecular Weight fraction, under the precipitation condiv tions used, has an average molecular'weight in the range of 50,000 to 300,000 as checked by the light scattering procedure. The lower molecular weight of the intermediate molecular weight fraction of reaction mixture A as compared to the intremediate molecular weight fraction of reaction B (as shown by the shift of the upper Division 63.)

portion of curve Ato the right) .'is probablyj'a' consealso thereto sucrose, the weight ratio of primer substance to totalsucrose substance being within the range of' 1:5 to 1:100.

2. The method of claim 1 in which the concentration of dextransucrase in the reaction mixture is within the range of 20 to 400 units per ml., where a unit of dextransucrase is defined as the amount of dextransucrase which will convert one mg. of sucrose to dextran in one hour at pH 5.0 and 30 C. p

3. The method of claim 1 in which the sucrose is added substantially all at one time, and the initial concentration is approximately 10 percent.

4. The method comprising interacting in an aqueous system dextransucrase, sucrose, and one of the group consisting of sucrodextran, maltodextran, and isomaltodextran, continuing the interaction to substantial decrease in sucrose concentration of the system, and recovering dextran of intermediate molecular weight from the reaction mixture by difierential precipitation with-a lower alkanol.

. 5. The-method of claim. 4 in which the primer sub-- stance is sucrodextran, ie., low molecular weight dextran produced in reaction mixtures with initially high sucrose concentrations.

6. The method of claim 4 in which the primer substance is maltodextran. I

7. The method of claim 4 in which the primer substance is isomaltodextran.

p. 8. The method comprising interacting in an aqueous system dextransucrase, sucrose, and, maltodextran of a 'molecular weight within the range of 5,000 to 30,000

maintaining. the system until the sucrose has decreased to a substantial amount, recovering dextran from the reaction mixtureby differential precipitation with a lower alkanol, separating. the dextran into an intermediate molecular weight fraction and a fraction having a molecular weight within the range of 5,000 to 30,000 and reintroducing vthe'latter intoan, aqueous system. comprising dextransucrase and sucrose to synthesize more dextran of intermediate molecular weight.

.9. The method of claim 8 in which. the ratio of .mal-

"todextran to initial sucrose concentration in the synthesis mixtures is within the range of 1:5 to 1:100. v

10,.The method of claim 1 in which the sucrose is added gradually so as to maintain a constant level. of sucrose in the system; r

1-1. The method of clam 1 in which the ratio of sucrose to primer-is maintained constant by the graduated addition of each and in which the ratio of sucrose to primer to jdextransucrase is also maintained constant.

I I References Cited in the file of thispatent Hehre et al.: Jour. Biol; Chem, 163, (1946), (Article pages '2'21233-), pages 221-222. QP-501-'J7. Scientific Library.

, Evans et al.: BacterialPolysaccharides, Sugar Research Foundation, Inc., New York, April 1947, Sci. Report Series No. 6, pages 211, 216,217, 228 to 230.

Hehreet al.: Jour. Bact., 55 (1948) pages 197-208. Pages specifically relied upon are 204205. -QR-1-J8.

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1. THE METHOD FOR PRODUCING DEXTRAN OF INTERMEDIATE MOLECULAR WEIGHT WHICH COMPRISES ADDING AS DEXTRAN PRIMER A GLUCOSE POLYMER CONTAINING PRINCIPALLY ALPHA LINKAGES BETWEEN THE GLUCOSYL GROUPS AND HAVING A MOLECULAR WEIGHT WITHIN THE RANGE OG 5,000 TO 30,000 TO DEXTRANSUCRASE ENZYME IN AQUEOUS SOLUTION AND ADDING ALSO THERETO SUCROSE THE WEIGHT RATIO OF PRIMER SUBSTANCE TO TOTAL SUCROSE SUBSTANCE BEING WITHIN THE RANGE OF 1:5 TO 1:100. 